中华根瘤菌L03几丁质酶纯化及其酶学性质研究

中华根瘤菌Sinorhizobium sp.菌株L03是1株能产生几丁质酶的生防细菌.采用90%饱和度硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等方法获得了菌株L03的几丁质酶.SDS-PAGE检测发现该酶已被纯化,其分子量约为41.3kD.该酶反应的最适温度为45℃;最适反应的溶液pH值为6;酶液在pH5～8条件下和40℃下分别保存1h,酶活力基本保持稳定;Mg2+和Ba2+能显著促进几丁质酶活性上升,而Zn2+、Cu2+和Fe3+等对酶活力有明显的抑制作用.功能分析结果显示,该几丁质酶对供试的几种病原真菌细胞壁都有明显的降解作用.     

拟康宁木霉T 51几丁质酶活性及内切几丁质酶基因克隆与分析

木霉是一类重要的生防真菌,木霉产生的几丁质酶在其生物防治的重寄生过程中起着重要作用。拟康宁木霉Trichoderma koningiopsis T-51是一株对番茄灰霉病有生防潜力的木霉菌株,本文测定了T-51菌株产生几丁质酶的活性,结果表明T-51在PDB中液体培养以及与灰霉病菌在PDA上对峙培养时产生的几丁质酶活性显著受到灰霉病菌的诱导。采用RACE技术首次克隆了拟康宁木霉T-51中一个几丁质酶基因Tkchit42,全长为1 817bp,包含4个外显子和3个内含子。预测该基因有一个1 275bp的开放阅读框,编码424个氨基酸,预测的蛋白总分子量为46.378kDa,预测的等电点(pI)为5.16,与T.koningii中42kDa内切几丁质酶的氨基酸序列相似性达到99%。     

绿色木霉几丁质酶基因Tvchi cDNA的克隆、原核表达与活性分析

 利用RT PCR方法从绿木霉ZBS 6中扩增了几丁质酶基因Tvchi,序列分析表明Tvchi 开放阅读框为1 293 bp,编码430个氨基酸残基,Blast 分析表明,与多种木霉18家族内切几丁质酶具有较高的同源性。将该基因克隆到原核表达载体pET 28a上,转化大肠杆菌BL21(DE3),经IPTG诱导,SDS PAGE和Western印迹分析表明成功的获得了47 kD 的融合蛋白。该融合蛋白经Ni NTA柱亲和纯化,获得了纯度较高的融合蛋白Tvchi。Tvchi最适酶活温度为37℃,最适酶活pH值为6.8。表达产物对小麦全蚀病、赤霉病、纹枯病病原菌显示出较好的抑菌活性。本研究结果将为进一步研究木霉几丁质酶的应用提供了基础。     

Pseudomonassp.1-7中甲基对硫磷水解酶基因的克隆及酶学性质研究

为了研究和分离有机磷降解酶及其编码基因,从农药厂污水处理池的活性污泥中分离出一株可以高效降解甲基对硫磷的细菌 1-7, 经16S rDNA鉴定为假单胞菌Pseudomonas sp.。通过构建基因组文库的方法克隆了 1-7 的甲基对硫磷水解酶基因 ophc3, 该基因全长为975 bp,编码324个氨基酸,其中前24个氨基酸残基可能为信号肽序列。将其在大肠杆菌中表达,并对重组甲基对硫磷水解酶(OPHC3)进行纯化和酶学性质的研究结果表明,其酶促反应最适pH值为8.0,在pH 6.0~10.0的范围内放置30 min酶的相对活性均在70%以上;最适反应温度为45 ℃,但该酶不耐高温,60 ℃下保温10 min,相对活性降至46.77%。     

寄生于南方根结线虫卵的长梗木霉几丁质酶基因TlChi46的克隆

为进一步筛选高效寄生线虫真菌和阐明其寄生线虫卵的机理,本研究从湖北省烟草南方根结线虫雌虫分离到1株具高效生防潜力的菌株HBF1。形态学、rDNA-ITS和翻译延长因子tef1-α序列分析鉴定该菌为长梗木霉菌Trichoderma longibrachiatum,其对南方根结线虫卵第10 d寄生率为80.45%。通过简并引物设计和RACE技术克隆其几丁质酶基因,分析该基因序列及与其他几丁质酶的同源性。该菌是1株可以产生几丁质酶的南方根结线虫卵寄生真菌,第10 d几丁质酶的活性达到高峰,为24.88μmol/h/mL。本研究首次从长梗木霉HBF1菌株中克隆到1个几丁质酶基因TlChi46,该基因DNA全长1 793 bp,含3个内含子和1个1 272 bp的开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.23。同源性比对表明和昆虫寄生菌几丁质酶的亲缘关系较远。从长梗木霉HBF1中克隆得到的几丁质酶基因编码的几丁质酶的功能域可供进一步研究,高效产几丁质酶并有效寄生南方根结线虫卵的长梗木霉对南方根结线虫具有良好的生防潜力。     

纤细齿梗孢丝氨酸蛋白酶cDNA克隆及其序列分析和表达

纤细齿梗孢Olpitrichum tenellum是一种寄生在轮枝镰孢菌Fusarium verticillioides上的活体营养菌寄生真菌。丝氨酸蛋白酶在菌寄生过程可能起到重要作用。根据丝状真菌丝氨酸蛋白酶的同源保守序列设计简并引物,通过RT-PCR及RACE的方法,克隆得到1845bp的全长cDNA片段。其可读框为1554bp（GenBank登录号为EU368754）,编码517个氨基酸的蛋白质。比对分析发现该蛋白是一种分泌型的丝氨酸蛋白酶,属于丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin,具有典型的信号肽-前肽-成熟肽的结构。将该基因可读框除去信号肽插入酵母表达载体pPIC9K,转化毕赤酵母Pichia pastoris GS115,在甲醇诱导下成功分泌出具有生物活性的重组蛋白酶,诱导120h后酶活性可达153U/ml。重组蛋白酶经DEAE-Sepharose层析进行纯化,SDS-PAGE分析其分子量为39kD。其反应的最适温度为60℃,最适pH为8。     

几丁质酶活性与大豆抗疫霉根腐病的关系

测定了大豆不同抗性品种接种大豆疫霉菌后的几丁质酶活性的变化情况。结果表明：未接种的不同大豆品种中的几丁质酶活性无明显区别。大豆疫霉菌侵染后不同抗性的大豆品种的几丁质酶的含量和活性均有不同程度的提高。抗病品种酶活性上升的速度比感病品种快,酶的高活性维持的时间较长。抗病品种在接种后24 h酶活性达到峰值,中感和感病品种在接种后48 h达到峰值。说明大豆疫霉菌可诱导几丁质酶产生,大豆品种的抗病性与几丁质酶的活性呈正相关关系。酶学特性的研究结果表明,酶反应的最适温度为45 ℃,最适pH为5。该酶在45 ℃以下,pH在4~6时酶活性稳定,超过60 ℃,pH超过7时酶活性丧失较快。Mn2+、Zn2+、Ba2+、Fe3+、Ca2+对酶活性有激活作用,Al3+、Ag+、Fe2+、K+、Mg2+、Cu2+、Hg2+对酶活性有抑制作用。     

粉红聚端孢菌胞外几丁质酶纯化、特性及抗菌活性

 粉红聚端孢菌(Trichothecium roseum)在SMCS中恒温摇床(200 r/min,28℃)上培养12 d,诱导产生了大量的胞外几丁质酶。培养滤液经(NH₄)₂SO₄分级沉淀、DEAE-Sepharose FF阴离子交换柱层析和CM-Sepharose FF阳离子交换柱层析获得了SDS-PAGE纯的胞外几丁质酶,分子量约为39k Da。几丁质酶最适作用温度为40℃,最适pH范围为4.0~7.0。抗菌活性显示,纯酶对供试病原菌都有不同程度的抑菌作用,其中对棉花黄萎菌的抑制作用最强。     

链霉菌769胆固醇氧化酶基因克隆、表达及生物学活性分析

胆固醇氧化酶(cholesterol oxidase,EC 1.1.3.6)是胆固醇降解代谢过程中的关键酶,在食品开发、医疗保健、临床检测、生物农药等方面具有广泛的应用价值。通过PCR方法从链霉菌Streptomyces ahygroscopicus 769的基因组中扩增得到769_ChOA基因(GenBank accession No. KF290994)。该基因全长为1578 bp,编码一个由525 aa组成的蛋白质,蛋白理论分子量为57 kDa,等电点为6.29。769_ChOA与来源于S. natalensis的胆固醇氧化酶基因的同源性为91%。构建了硫氧还蛋白－769_ChOA融合的表达载体pET32a::769_ChOA,以Escherichia coli Origami B(DE3)为表达宿主,获得了能可溶性表达的胆固醇氧化酶重组菌。经Ni亲和层析纯化得到的酶蛋白比活力为5.90 U/mg。酶学性质分析表明,该酶对胆固醇的催化活性最高,对测定的7种胆固醇类似物也都具有一定的催化能力。酶的最适温度和pH范围为20～40 ℃和pH 6.0～8.5,其活性均高于80%,且在30 ℃和pH 7.0时表现出最大的活性,酶活分别达到6.83和6.44 U/mg。酶在低于40 ℃和弱碱性条件下具有很好的稳定性。该酶对鳞翅目害虫亚洲玉米螟Ostrinia furnacalis(Guenée)和水稻二化螟Chilo suppressalis(Walker)幼虫具有很强的杀虫活性,半致死浓度分别为27.4和17.1 mg/mL。     

暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

cDNA文库免疫筛选到编码暗黑鳃金龟幼虫几丁质脱乙酰酶HpCDA5基因,序列分析表明HpCDA5含有1个几丁质脱乙酰酶结构域,属于Group V类CDA蛋白。构建重组杆状病毒表达载体pFastBac-HpCDA5,转染昆虫细胞sf9,Western blot分析表明HpCDA5在昆虫细胞sf9中成功表达42 kDa的蛋白。利用qRT-PCR方法分析HpCDA5基因组织表达,结果显示HpCDA5基因在中肠中表达最高,为中肠特异表达蛋白。几丁质结合活性表明HpCDA5蛋白只能被强洗脱剂洗脱,具有很强的几丁质结合活性。本研究通过对暗黑鳃金龟几丁质脱乙酰酶HpCDA5的生化特性研究,为进一步明确HpCDA5的生理功能提供理论依据,并为以HpCDA5蛋白为靶标的暗黑鳃金龟生物防治提供支撑。     

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus

ABSTRACT The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.     

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

Purification of an Elicitor-Inducible Antifungal Chitinase from Suspension-Cultured Rice Cells

Suspension-cultured rice cells showed an appreciable amount of chitinase activity when the cultured cells were treated with an elicitor isolated fromRhizoctonia solani, the rice sheath blight pathogen. A fivefold increase in chitinase activity was observed 24 h after elicitor treatment. The elicitor-inducible chitinase was purified by ammonium sulfate fractionation, chitin affinity chromatography and gel filtration. Its molecular weight is 35 kDa and it has an isoelectric point of 8.3. The 35 kDa basic chitinase inhibited mycelial growth ofR. solani in vitro. Morphological changes appeared within 1 h following exposure of mycelium to the chitinase. The hyphal tip showed marked swelling and subsequently lysis was also observed.     

产生几丁质酶的食线虫真菌绿粘帚霉Gliocladium virens CFCC80915对南方根结线虫卵孵化的影响

 根结线虫卵壳主要由几丁质和蛋白质构成。寄生根结线虫卵或产毒真菌产生几丁质酶特性是评价食线虫真菌生物防治潜力的重要生化指标之一。利用还原糖法和pNP法分别测定绿粘帚霉(Gliocladium virens)的几丁质酶的内切酶和外切酶活性。第14d内切酶活性达到最高值,其酶活为53.1μmol/h mL;外切酶活性第26d达到高峰,其酶活为0.432μmol/h mL。利用活性染色电泳测其几丁质酶的分子量分别为75.8kDa、42.8kDa和39.6kDa。表明筛选到的绿粘帚霉CFCC80915菌株具有较高产生几丁质酶的活性。绿粘帚霉12d的培养滤液对根结线虫卵孵化7d后的抑制率达到92.9%。显微观察线虫卵壳变形和破坏情况的结果表明,绿粘帚霉CFCC80915产生的几丁质酶可以引起根结线虫卵壳的裂解,抑制根结线虫卵的孵化。     

Differential induction of pathogenesis-related proteins in tomato byAlternaria solaniand the association of a basic chitinase isozyme with resistance

Accumulation of pathogenesis-related proteins is thought to play a role in pathogen-induced plant defense responses. Although early accumulation of hydrolytic enzymes such as chitinase and β-1,3-glucanase has been associated previously with genetically-inherited and induced systemic resistance, their role in resistance in tomato(Lycopersicon esculentum)to the phytopathogenic fungusAlternaria solaniis not yet understood. Here we describe the accumulation patterns of specific isozymes of pathogenesis-related proteins in the resistant tomato genotypes 71B2, NC EBR-1, NC EBR-2 and the susceptible cultivar Piedmont. Western blot analysis demonstrated that four isozymes of chitinase (26, 27, 30, and 32kDa) were induced in all genotypes upon challenge withA. solani,but only resistant lines had significantly higher constitutive levels of the 30kDa isozyme as well as total chitinase activity. In addition, the 30kDa chitinase isozyme was found to accumulate to significantly higher levels in resistant lines during pathogenesis than the susceptible genotype. Two isozymes of β-1,3-glucanase (33 and 35kDa) were detected in all genotypes, but a slightly higher constitutive level was detectable in all resistant lines when compared to the susceptible. Similar accumulation patterns of these isozymes were observed in all genotypes during the course of pathogenesis. Purified preparations of acidic and basic tomato chitinase and β-1,3-glucanase isozymes were tested for their antifungal activity againstA. solani in vitro.Results presented in this study indicate that only basic isozymes of chitinase and β-1,3-glucanase were inhibitory toA. solaniwhereas, no inhibitory activity was observed with the acidic isozymes. The results of this study suggest that a higher constitutive level of chitinase and β-1,3-glucanase and the induction pattern of a 30kDa chitinase isozyme in early blight resistant breeding lines is related to genetically-inherited resistance of tomato toA. solani.